Heparin and its derivatives, low molecular weight heparins (LMWHs) belong to one category of glycosaminoglycan (GAG). They are important anticoagulant drugs resulting from their anticoagulation functions. In order to reduce the risk of side effects, such as bleeding, osteoporosis, thrombocytopenia, and promote the bioavailability, LMWHs are widely utilized as new anticoagulant instead of heparin. LMWHs are manufactured by either enzymatical or chemical degradations making each kind of LMWH possessing its own special structures. Enoxaparin is a LMWH manufactured via alkaline depolymerization of benzyl ester of heparin. Its major non-reducing ends (NREs) are unsaturated uronic acid residues after chemical modification, while saturated uronic acid and amino sugar residues from the parent heparin are also existed. The major reducing ends (RE) are amino sugars which containing 15-25% of 1,6-anhydro structure, also there are a small amount of uronic acid residues and linkage region at the RE. Dalteparin is a kind of LMWH manufactured via nitrous acid degradation. Its major NREs are saturated uronic acid residues, while major REs is mannitol and a small amount of linkage region. In addition to these special ending structures, the backbone also varies during the manufacturing. All above make the analysis of complete degradation products of LMWHs a big challenge. Usually, there are two strategies for heparin analysis, top-down and bottom-up. Bottom-up analysis is the first choice for degradation product analysis. Heparin products generally enzymatically degraded to complete degradation products, and then are analyzed by capillary electrophoresis, high performance liquid chromatography (HPLC) and HPLC tandem mass spectrometry. These conventional methods were mainly focus on the 8 natural disaccharides and only a part of the special structures, not one can perform a comprehensive identification and quantitation analysis on complete degradation products derived from LMWHs. Take enoxaparin for instance, the original ending structures are not able to identified as the newly generated ending structures of building blocks during enzymatic digestion are identical to the original ones. However, these special structures with extremely low content usually contain important structural information related to the quality and safety of drugs. In other words, the characterization of these building blocks are essential.
In addition to the 8 natural disaccharides, the complete degradation products consist of 3-O-sulfated tetrasaccharides which related to the anticoagulation function, trisaccharides generated by peeling reaction, saturated NREs from parent heparin, N-unsubstituted disaccharides and galacturonic acid disaccharides from chemical modification and C-sulfated disaccharide and epoxide structures from molecular rearrangement. Moreover, there are some characteristic structures for different LMWHs, for example, 1,6-anhydro structures in enoxaparin and its original NRE and RE structures, 2,5-anhydro mannitol. No reported methods are able to cover all these building blocks, nevertheless, the analysis of these ending structures and special structures are indispensable for development of LMWH generic drugs, production control and safety control. Moreover, as some of the original ending structures, like enoxaparin, are identical to that of newly generated ending structures after enzymatical digestion, conventional samples preparation strategies are not capable for comprehensive characterization of enoxaparin.